Introduction: Poly (ADP‐ribose) polymerase (PARP) inhibitor has been widely used in ovarian cancer patients carrying BRCA mutations. However, resistance to PARP inhibitor is present in some patients, and no effective treatment is available for these patients. TH‐302 is a hypoxia‐activated prodrug, which releases the bis‐DNA alkylator bromo‐isophosphoramide mustard (Br‐IPM) under hypoxic condition. The present study aims to determine whether TH‐302 is effective in treating PARP inhibitor resistance.Methods: The in vitro cytotoxicity of TH‐302 was assessed by short‐term proliferation assay (50% inhibitory concentration, IC50) or long‐term clonogenic assay (90% inhibitory concentration, IC90) under various oxygen concentrations. In vivo efficacy of TH‐302 was assessed in PARP inhibitor resistance, partially responsive and sensitive patient‐derived xenograft (PDX) or cell line–derived xenograft (CDX) models. Antitumor activity via homologous recombination (HR) pathway for TH‐302 was evaluated using DLD1 BRCA2 knockout cell line and BRCA/RAD51D deleterious mutant PDX/CDX models. Breaks of double‐strand DNA and hypoxia fraction in tumors were determined by gamma histone 2AX (γH2AX) and pimonidazole immunohistochemistry in H460 CDX model following treatment.Results: Cytotoxicity was significantly enhanced under hypoxia in 12 human cancer cell lines including four ovarian cancer cell lines. The cytotoxicity was 70 times higher in human colon cancer cell line with BRCA2 knock out compared to wild type under hypoxia following TH‐302 treatment. γH2AX staining revealed that the cytotoxicity of TH‐302 was associated with DNA damage. In addition, administration of TH‐302 with olaparib led to better antitumor activities than either single drug/prodrug in olaparib‐resistant PDX models.Conclusion: TH‐302 exhibits hypoxia‐dependent cytotoxicity across a wide range of human cancer cell lines, and may be a drug candidate to treat ovarian cancer, bladder cancer, and pancreatic cancer with HR deficiencies with or without resistance to PARP inhibitor. TH‐302 may be effective in recurrent epithelial ovarian cancer (EOC) with homologous recombination deficiency (HRD) or in EOC patients resistant to PARP inhibitors.